JP2006280234A - Method for proliferating or differentiating cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for proliferating or differentiating cells, by which the cells can efficiently be proliferated or differentiated in a simple operation without simultaneously co-culturing two or more kinds of cells. <P>SOLUTION: This method for proliferating or differentiating the cells is characterized by culturing the second kind of the cells in the presence of the culture supernatant of the first kind of cells to promote the proliferation or differentiation of the second kind of the cells. Especially, the method for proliferating or differentiating the cells comprises culturing mesenchymal cells in the presence of the culture supernatant of epithelial cells. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for cell proliferation or differentiation. More specifically, the present invention relates to a method for controlling the growth or differentiation of cells of another species using the culture supernatant of one cell.

  In order to realize cell medicine and regenerative medicine, it is necessary to establish a technique for efficiently controlling cell proliferation and differentiation. In order to efficiently proliferate and differentiate cells using conventional cell culture methods, culture is performed using cytokines such as fibroblast growth factor (FGF) and endothelial growth factor (EGF), and bioactive agents such as dexamethasone. However, there is a limit to cell control with a single factor, and there are concerns about side effects and the like when using artificially synthesized physiologically active substances. Therefore, in order to solve such problems, there is a need for a technique that does not place an excessive load on cells and efficiently controls cell proliferation / differentiation.

  Patent Document 1 discloses, as a cell culture technique using a culture supernatant, a procedure for adding a self-tissue extract to a serum-free medium that does not contain animal-derived components other than autologous cells, and culturing human cells. A method of continuing cell culture using a culture supernatant containing a substance secreted from the human cells in the course of the above, a cell cultured by the method containing components derived from animals other than self A method for separating and subcultured in a serum-free medium and a method for cryopreserving cells cultured by these methods is described. The technique described in Patent Document 1 improves the cell growth ability in a serum-free medium by adding a culture supernatant as an additional component of the serum-free medium, and is intended to control cell growth and differentiation is not.

  Non-Patent Document 1 describes that cell calcification and differentiation induction are performed by mixed culture of epithelial and mesenchymal cells. Furthermore, Non-Patent Document 2 describes that the synthesis of alkaline phosphatase, which is a marker for differentiation into odontoblasts, is increased in dental pulp cells co-cultured with rabbit incisor-derived epithelial cells. However, in these methods, there is a problem that it is necessary to simultaneously control a plurality of types of cells in a single container, and there is a possibility that extra cells may be mixed when applied clinically.

JP 2004-173563 A Ishizeki, K., Acta Anat Nippon 71: 294-307 (1996) Hideki Shiba et al., Cell Biology International 27 (2003) 815-823

  The present invention has been made to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a method capable of efficiently proliferating and differentiating cells with a simple operation without performing co-culture in which two or more types of cells are cultured simultaneously.

  As a result of intensive studies to solve the above problems, the present inventors have cultivated mesenchymal cells in the presence of a culture supernatant of certain epithelial cells, thereby proliferating and differentiating mesenchymal cells. The present invention has been completed.

  That is, according to the present invention, the growth or differentiation of the second type of cells is promoted by culturing the second type of cells in the presence of the culture supernatant of the first type of cells. A method for controlling cell proliferation or differentiation is provided.

Preferably, mesenchymal cells are cultured in the presence of a culture supernatant of epithelial cells.
Preferably, the mesenchymal cells are cultured in the presence of an epithelial cell culture supernatant on a carrier.
Preferably, as an epithelial cell, an inner enamel epithelial cell, an outer enamel epithelial cell, an enamel medullary cell, an intermediate layer cell, an enamel blast, a Marasse epithelial cell, an oral mucosal epithelial cell, an epithelial cell or a progenitor cell thereof Is used.
Preferably, as mesenchymal cells, tooth germ mesenchymal cells, odontoblasts, dental pulp cells, tooth papilla cells, dental follicle cells, cementoblasts, osteoblasts or their precursor cells, or mesenchymal stem cells Is used.

According to one embodiment of the present invention, there is provided a method for promoting proliferation of mesenchymal stem cells, comprising culturing mesenchymal stem cells in the presence of a culture supernatant of oral mucosal epithelial cells.
According to another embodiment of the present invention, there is provided a method for promoting differentiation of tooth germ mesenchymal cells, comprising culturing tooth germ mesenchymal cells in the presence of a culture supernatant of tooth germ epithelial cells. .

According to another aspect of the present invention, proliferated or differentiated cells obtained by the above-described method of the present invention are provided.
According to still another aspect of the present invention, a method for regenerating a tooth germ using mesenchymal cells proliferated or differentiated by the above-described method of the present invention is provided.

  The present invention utilizes the effect of a physiologically active substance contained in a culture supernatant obtained at the time of cell culture medium exchange, and uses this culture supernatant for culturing another cell, thereby proliferating the cell.・ Technology that promotes differentiation. As shown in the examples of the present invention described below, desired cells can be proliferated and differentiated by changing the combination of cells from which the culture supernatant is derived and cells cultured using the supernatant. . Since the present invention is a technique that applies the physiologically active action inherent to living bodies, cells can be efficiently proliferated and differentiated without placing an unnatural burden on the cells. Also, in situations where it is difficult to use factors derived from other families, such as transplantation medicine, cell therapy, and regenerative medicine, it is possible to use only patient-derived tissues and cells by combining patients-derived cells without using other factors. A system capable of freely controlling cell proliferation / differentiation can be constructed. In addition, since the cell culture in the present invention is a single type of cell culture, the control of the cells is relatively simple, and there is no risk of extra cells being mixed in clinical applications. Have advantages.

Hereinafter, embodiments of the present invention will be described in detail.
The method for controlling cell proliferation or differentiation according to the present invention comprises culturing a second type of cell in the presence of a culture supernatant of the first type of cell to thereby proliferate or differentiate the second type of cell. It is a method characterized by promoting. According to a preferred embodiment, mesenchymal cells can be cultured in the presence of a culture supernatant of epithelial cells. According to another aspect of the present invention, epithelial cells can be cultured in the presence of a culture supernatant of mesenchymal cells.

  The present invention is a technique utilizing the fact that factors associated with in vivo epithelial-mesenchymal interaction are secreted into the culture supernatant when cells are cultured. In living organisms, the interaction between epithelial and mesenchymal cells is important, and for the development of organs and tissues in the living body, or to maintain their morphology and function, proliferation and differentiation from epithelial and mesenchymal cells at each site The regulatory factor is released and the organs and tissues are generated and maintained.

  The present invention makes use of such biological characteristics and expects the effect of a physiologically active substance contained in the culture supernatant that is produced when the culture medium is changed in the cell culture. It is a technology that promotes cell proliferation and differentiation by using it (FIG. 1).

  In the present invention, for example, epithelial cells can be used as cells for collecting the culture supernatant. The epithelial cell used in the present invention is not particularly limited as long as it is an epithelial cell, but is preferably an inner enamel epithelial cell, an outer enamel epithelial cell, an enamel medullary cell, an intermediate layer cell, an enamel blast, a marasse cell. Epithelial remnant cells, oral mucosal epithelial cells, epithelial cells, epidermal cells, or progenitor cells thereof. These cells may be cultured as a single cell composed of one type of epithelial cell, or may be cultured as a cell mixture composed of two or more types of epithelial cells.

  As cells for promoting proliferation or differentiation in the present invention, for example, mesenchymal cells can be used. The mesenchymal cell is not particularly limited as long as it is a mesenchymal cell, but is preferably a tooth germ mesenchymal cell, odontoblast, pulp cell, tooth papilla cell, dental follicle cell, cementoblast , Osteoblasts or their progenitor cells, or mesenchymal stem cells. These cells may be cultured as a single cell composed of one type of mesenchymal cell, or may be cultured as a cell mixture composed of two or more types of mesenchymal cells.

  Epithelial cells can be collected from mammals (eg, humans, pigs, etc.) tooth germ, periodontal ligament (marasse epithelial residue), oral mucosa, adherent epithelium, skin and the like by known methods. For example, in the case of epithelial cells such as inner enamel epithelial cells, outer enamel epithelial cells, enamel medullary cells, intermediate layer cells, and enamel blasts, they can be collected from the mandible of mammals (eg, humans, pigs, etc.) . The impacted teeth are aseptically removed and stored in a suitable storage solution such as Hanks balanced salt solution (HBSS) solution. The calcified portion in the tooth is removed, the tissue is cut into small pieces with a scalpel, and the tissue is washed with an HBSS solution or the like. Subsequently, it is preferable to perform enzyme treatment of the tissue using collagenase and dispase. After the enzyme treatment, the cells can be collected by pipetting and centrifugation. When the obtained cells are cultured using, for example, MCDB153 (kyokuto Co.) as a medium, mesenchymal cells in the tooth germ are lost, and only epithelial cells can be obtained.

  In the case of oral mucosal epithelial cells, it can be obtained by treating oral mucosa collected from a human with dispase, then peeling off the epithelial portion and treating with trypsin.

  The culture supernatant of epithelial cells can be collected by a conventional method known to those skilled in the art. For example, the culture supernatant can be collected at the time of medium exchange during the culture of epithelial cells, and the collected culture supernatant can be filtered to remove floating cells and impurities before use. .

  Mesenchymal cells can be collected from tooth germs, dental pulp, alveolar bone, bone marrow, and the like of mammals (eg, humans, pigs, etc.) by known methods. For example, mesenchymal cells in a tooth germ can be collected from the mandible of a mammal (eg, human, pig, etc.). The impacted teeth are aseptically removed and stored in a suitable storage solution such as PBS solution or HBSS solution. The calcified portion in the tooth is removed, the tissue is cut into small pieces with a scalpel, and the tissue is washed with PBS solution or HBSS solution. Subsequently, it is preferable to perform enzyme treatment of the tissue using collagenase and dispase. After the enzyme treatment, the cells can be collected by pipetting and centrifugation. When the obtained cells are subcultured using Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% antibiotics, the epithelial cells in the tooth germ are lost and mesenchymal Only cells can be obtained.

In addition, the removal of the dental pulp from the tooth can be performed according to the method described in, for example, About I., Other Experimental cell research.258.33-41,2000. Mesenchymal cells can be obtained by transferring aseptically collected dental pulp to a petri dish and culturing it in a medium.
Furthermore, according to a known method, mesenchymal stem cells can be obtained by performing bone marrow puncture from the iliac or the like to collect and culture the bone marrow.

  The cells cultured according to the method of the present invention (for example, mesenchymal cells) can be used for cell medicine and regenerative medicine because their proliferation or differentiation is promoted. For example, it is possible to form odontoblasts or tooth germs by proliferating and differentiating tooth germ mesenchymal cells, which are used for treatment of the patient by transplanting it to a dental patient. In this case, from the viewpoint of biocompatibility associated with transplantation, the mesenchymal cells used for the culture are preferably the own cells derived from the patient, but the same type (other family) cells may also be used. Is possible.

  It is known that teeth are formed through five stages from development to maturity. The first stage is called the initiation stage, where epithelial and mesenchymal tissues are induced in the basement membrane. The second period is called Bud stage, and an enamel device is made. The third stage is called Cap stage, where the tooth papilla is formed and the tooth germ is formed. The fourth stage, called the Bell stage, begins to differentiate from the tooth germ into cells that form enamel and from the tooth papilla into cells that form dentin and pulp. The fifth stage is called the "Maturation stage" and differentiates into tissues that make up the tooth, such as enamel, dentin, and pulp. Of the epithelial cells and mesenchymal cells used in the present invention, cells at a suitable time can be collected and used. In cases where no tooth germ is present, the pulp can be removed from the root and cells can be isolated and collected.

Culture of epithelial cells and mesenchymal cells is carried out using normal serum-containing medium used for animal cell culture under normal animal cell culture conditions (for example, a temperature of room temperature to 37 ° C .; 5 to 10% CO _2. Under the incubator). In addition, the epithelial cells can be cultured using a serum-free medium, or in the presence of feeder cells such as fibroblasts.

  In the present invention, cells may be cultured on a carrier or may be cultured without a carrier. The use of a carrier is useful for forming tooth germs from cells. As the carrier, a carrier that can endure the time required for the formation of a tooth germ and is rapidly absorbed thereafter is preferable. That is, it is preferable to use a carrier made of a material having an appropriate absorption rate and characteristics in a living body such as subcutaneous, stomach omentum, or jawbone, and having a high affinity for cells.

  The material of the carrier is not particularly limited as long as it satisfies the above characteristics. For example, polyglycolic acid (PGA), poly (DL-lactide-co-glycoside) (PLGA), polylactic acid (PLLA) Synthetic polymer materials such as polycaprolactone, protein materials such as collagen, gelatin, and fibrin, or natural materials such as hyaluronic acid and salts thereof, alginic acid and salts thereof, dentin, and coral can also be used. Furthermore, inorganic materials such as tricalcium phosphate (β-TCP) can also be used.

  PGA can be purchased from Albany International Research Co., for example, and PLGA can be purchased from Sigma. In the case of PGA, since the absorption rate is fast, poly (DL-lactide) (PLLA) can be coated on the surface to delay the absorption period. Further, when a synthetic material such as PGA, PLLA, PLGA or polycaprolactone is used, it can be used by coating a collagen solution or fibronectin solution on the surface in order to enhance cell adhesion and proliferation.

  Examples of the form of the carrier include a mesh form, a sponge form, a gel form, and a non-woven form.

  The carrier is preferably processed into a shape in which cells can be easily transplanted, and is preferably a plate-like or spherical porous body or hollow, with one end being opened and a blood vessel easily entering from the periphery.

  The carrier is preferably prepared in a form suitable for the purpose. For this purpose, a mold is obtained using an impression material after a desired form is made of a resin. Thereafter, the desired form can be reproduced by taking out the resin mold and pouring the synthetic material constituting the carrier.

  In the method of the present invention, after culturing mesenchymal cells in the presence of epithelial cell culture supernatant, the mesenchymal cells are transplanted into a transplanted animal, and dentin and tooth germ are transplanted in the transplanted animal body. The cells may be regenerated, or the mesenchymal cells may be directly transplanted into the patient. The carrier used for culturing the cells can also be transplanted together with the cells into the body of the transplanted animal.

Although the kind of transplant animal is not specifically limited, Preferably it is a mammal, For example, rodent animals, such as a rat (hairless rat etc.), a rabbit, or a mouse | mouth, can be used. The site for transplantation is preferably a site that can easily supply factors necessary for bone formation, and specifically, a site where blood flow is abundant is preferable, for example, an intraperitoneal stomach omentum is particularly preferable. By transplanting into such a site, cell growth can be promoted, and bone formation can be accelerated.
The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1: Proliferation of mesenchymal stem cells using culture supernatant of oral mucosal epithelial cells (method)
Rat oral mucosal epithelium was cultured in maintenance medium, and the oral mucosal epithelial medium that reached confluence was replaced. Thereafter, the medium was changed every three days, and the culture supernatant produced during the medium exchange was collected. The collected culture supernatant was subjected to filter filtration to completely remove floating cells and impurities.

  The culture supernatant collected by the above method is diluted with an arbitrary amount of DMEM, and the culture supernatant is used as a medium to culture mesenchymal stem cells (MSC). The effect of the culture supernatant on the proliferation ability of MSC It was investigated.

(result)
An initial number (1 × 10 ⁴ ) is seeded on a 6-well plate, DMEM medium, DMEM: maintenance medium = 1: 1, DMEM: culture supernatant = 1: 1, DMEM: culture supernatant = 3: 1, DMEM = As a result of examining the culture medium of culture supernatant = 7: 1, MSC cultured in a medium having a mixing ratio of DMEM: culture supernatant = 7: 1 showed high cell proliferation ability (FIG. 2). In addition, no decrease in cell morphology or growth rate was observed, suggesting that this culture supernatant has the property of causing cell growth on MSC.

Example 2: Differentiation of tooth germ mesenchymal cells using culture supernatant of tooth germ epithelial cells (method)
Porcine tooth germ epithelial cells isolated from pig tooth germ using dispase were cultured in KGM medium, and the medium of congested tooth germ epithelium was replaced. Thereafter, the medium was changed every three days, and the culture supernatant produced during the medium exchange was collected. The collected culture supernatant was subjected to filter filtration to completely remove floating cells and impurities.

  The culture supernatant collected by the above method is diluted with an arbitrary amount of DMEM, and the embryonic mesenchymal cells are cultured using this culture supernatant as a culture medium. The effect of the supernatant was examined.

(result)
An initial number (1 × 10 ⁴ ) is seeded on a 12-well plate, DMEM medium, DMEM: KGM medium = 1: 1, DMEM: KGM medium = 1: 3, DMEM: KGM medium = 3: 1, DMEM = on top of culture Kiyoshi = 1: 1, DMEM: culture supernatant = 1: 3, DMEM: culture supernatant = 3: 1, examination of tooth germ epithelial supernatant medium, tooth germ mesenchyme using tooth germ epithelial supernatant The system cells showed almost no cell proliferation, but very high ALP activity (FIG. 3). Therefore, in the tooth germ mesenchymal cells cultured using the culture supernatant, cell proliferation hardly occurred, while high ALP activity was exhibited. It was suggested that the cells have the property of causing cell differentiation.

FIG. 1 shows a cell culture technique using a culture supernatant according to the present invention. FIG. 2 shows the cell proliferation action by the culture supernatant of oral mucosal epithelial cells. A shows the change in the number of cells of MSC cultured with the culture supernatant of oral mucosal epithelial cells. B shows the increase rate based on the initial cell number. FIG. 3 shows the cell differentiation action by the culture supernatant of tooth germ epithelial cells. A shows changes in the number of tooth germ mesenchymal cells cultured in the culture supernatant of tooth germ epithelial cells. B shows changes in ALP activity of tooth germ mesenchymal cells cultured with tooth germ epithelial cells.

Claims (5)

Proliferation or differentiation of a cell characterized by promoting the proliferation or differentiation of the second type of cells by culturing the second type of cells in the presence of the culture supernatant of the first type of cells Control method. The method according to claim 1, wherein the mesenchymal cells are cultured in the presence of a culture supernatant of epithelial cells. The method according to claim 2, wherein the mesenchymal cells are cultured on a carrier in the presence of a culture supernatant of epithelial cells. As the epithelial cells, inner enamel epithelial cells, outer enamel epithelial cells, enamel medullary cells, intermediate layer cells, enamel blasts, Marasses epithelial cells, oral mucosal epithelial cells, epithelial cells or their progenitor cells are used. The method according to claim 2 or 3. As mesenchymal cells, tooth germ mesenchymal cells, odontoblasts, dental pulp cells, tooth papilla cells, dental follicle cells, cementoblasts, osteoblasts or their precursor cells, or mesenchymal stem cells are used. A method according to any one of claims 2 to 4.

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